PXD007991

PXD007991 is an original dataset announced via ProteomeXchange.

Title	Molecular Basis of the mechanisms controlling MASTL - Proteomics analysis of MASTL substrate proteome and phosphorylation state
Description	As part of our study on human MASTL (microtubule-associated serine/threonine kinase-like), we used mass spectrometry (MS)-based proteomics in the aim to unravel novel aspects about MASTL activation, regulation and potential substrates. Specifically, we performed a phosphoproteomics-based kinase assay (essentially as in Xue et al. 2014) to identify MASTL substrates and analyzed different MASTL constructs to generate a “phosphorylation map” of the MASTL protein. siKALIP (Stable Isotope Labelled Kinase Assay-Linked Phosphoproteomics) Three MASTL variants, which we termed MASTL (full-length), MASTL450 (short) and Bonsai (truncated), were expressed in HEK293 cells and isolated using a tandem Strep and His-tag affinity purifications. For the in vitro kinase assay we used a dephosphorylated cell extract from interphase HEK293 cells to perform a slightly modified version of the siKALIP (described in Xue et al. 2014). Samples included an untreated basal sample, a dephosphorylated sample and samples for each of the three MASTL constructs. For label-free quantification, reactions were performed in quadruplicates for each MASTL construct. Explanation for MS raw files with MASTL construct included: FL: MASTL full-length. 450short: Shorter (consisting of the first 450 amino acid residues) MASTL. Bonsai: Truncated MASTL (consisting of amino acid residues 35-112, 113-180 and 728-879). MASTL phosphorylation map To analyse the phosphorylation state of MASTL we used isolated protein from an interphase HEK293 cell culture, either alone or incubated with mitotic extracts from HEK293 cells. Samples of different MASTL constructs were used for in-gel digestion and analysed by MS. Explanation for MS raw files with MASTL construct included: FL: MASTL full-length (“INACTIVE” indicates kinase-dead, “Dephos” indicates dephosphorylated FL protein, “Mit” indicates incubated with mitotic extracts). 450: Shorter (450 amino acids) MASTL (“KD” indicates kinase-dead). BONSAI: Truncated MASTL (“KD” indicates kinase-dead).
HostingRepository	PRIDE
AnnounceDate	2024-10-22
AnnouncementXML	Submission_2024-10-22_04:16:34.660.xml
DigitalObjectIdentifier
ReviewLevel	Peer-reviewed dataset
DatasetOrigin	Original dataset
RepositorySupport	Unsupported dataset by repository
PrimarySubmitter	Anna-Kathrine Pedersen
SpeciesList	scientific name: Homo sapiens (Human); NCBI TaxID: 9606;
ModificationList	phosphorylated residue
Instrument	Q Exactive HF

Revision	Datetime	Status	ChangeLog Entry
0	2017-10-18 04:09:11	ID requested
1	2020-01-09 10:38:11	announced
⏵ 2	2024-10-22 04:16:35	announced	2024-10-22: Updated project metadata.

Hermida D, Mortuza GB, Pedersen AK, Pozdnyakova I, Nguyen TTTN, Maroto M, Williamson M, Ebersole T, Cazzamali G, Rand K, Olsen JV, Malumbres M, Montoya G, Molecular Basis of the Mechanisms Controlling MASTL. Mol Cell Proteomics, 19(2):326-343(2020) [pubmed]

10.1074/mcp.ra119.001879;

curator keyword: Biological

submitter keyword: Human, LC-MSMS, MASTL

Guillermo Montoya
contact affiliation	Macromolecular Crystallography Group, NNF Center for Protein Research, Faculty of Health and Medical Sciences, University of Copenhagen, Blegdamsvej 3B, Copenhagen, 2200, Denmark
contact email	guillermo.montoya@cpr.ku.dk
lab head
Anna-Kathrine Pedersen
contact affiliation	NNF Center for Protein Research, University of Copenhagen
contact email	anna-kathrine.pedersen@cpr.ku.dk
dataset submitter

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PRIDE project URI

• PRIDE
  1. PXD007991
     1. Label: PRIDE project
     2. Name: Molecular Basis of the mechanisms controlling MASTL - Proteomics analysis of MASTL substrate proteome and phosphorylation state